The purpose of this tutorial is to provide a step-by-step guide to
isolate nuclei from flash-frozen Kidney synthetic
assembloids for single-nucleus RNA sequencing.
Nuclei Isolation is the first step in many novel techniques including
snNRAseq, single-nucleus assay for transposase-accessible chromatin
sequencing, epigenomic analysis. Although based off of the 10X Genetics
Chromium Nuclei Isolation Protocol, this tutorial is specifically for
the use of Kidney synthetic assembloids for snRNAseq and includes
alterations to maximize yield and quality. This tutorial will also
include a guide on how to assess and grade the quality of the isolated
nuclei as well as next steps after the initial isolation.
Critical to pellet the Assembloids prior to flash freezing in the
sample tubes supplied by 10X chromium so it fits the pestle.
Work quickly helps with maintaining the quality of the nuclei.
Depending on the starting material and step that your working on, it
might be challenging to visualize the nuclei (especially after the
debris removal step), so make sure to know the orientation of the tubes
in the centrifuge.
Waiting 15 minutes at the end of the protoco help for the damaged
nuclei to clump together so that mostly high-quality nuclei remain in
the sample and are resuspended.
Key Modifications
Multiple modifications have been made to optimize this protocol for
the small tissue of assembloids.
Decreased volume of Lysis Buffer needed
(100 µL)
Decreased number of pestle twists to dissociate
tissue
We found that 5 was suffecient but **vissually
confirmed the solution was absense of large chunks
Skip the step to run solution through a column to
remove large debris
Perform ALL centrifugations in a Round
Bottom FACS tube and a Swinging Bucket Rotor
Decreased Wash Buffer volumes to 500 µL
Final resuspension in 100 µL
Consumables
List of Consumables
Action
Item
10X PN
Preparation & Handling
Storage
Place on Ice
Lysis Reagent
2000558
Vortex, verify no precipitate, and centrifuge briefly.
4°C
Surfactant A
2000559
Vortex, verify no precipitate and centrifuge briefly.
4°C
Debris Removal Reagent
2000560
Vortex, verify no precipitate and centrifuge briefly.
4°C
Reducing Agent B
2000087
Thaw to RT, vortex, verify no precipitate, and centrifuge briefly.
−20°C
RNase Inhibitor
2000565
Centrifuge briefly.
−20°C
Nuclei Isolation Consumables:
Nuclei Isolation Column
2000562
Pre-chill both on ice
Ambient
Collection Tube
2000563
Pre-chill both on ice
Ambient
Nuclease-free Water
—
See Buffer Preparation
Ambient
1X PBS
—
See Buffer Preparation
Ambient
10% BSA
—
See Buffer Preparation
4°C
Round Bottom FACS tubes
Corning (352003)
Ambient
Place on Dry Ice
Frozen Tissue Sample
—
Thaw on ice
Liquid Nitrogen (long-term) or −80°C (short-term)
1. Start with the Wash and Resuspension
Buffer, which is composed of the 1X PBS and 10%
BSA solutions that are not included in the kit.
Generally, make a stock of 100 mL of each and keep chilled at 4°C. Refer
to the table above and the instructions for components and amounts.
1X PBS: Dilute 10 mL into 90 mL of Ultra-Pure
Water. Invert to mix thoroughly. 10% BSA: 10
grams of powder BSA to 100 mL of Ultra-Pure Water. Mix gently by
swirling to prevent excess frothing.
2. Next, retrieve all components of the 10X
Nuclei Isolation Kit, which should include the Lysis Reagent,
Debris Removal Reagent, Reducing Agent B, Surfactant A, and RNAse
Inhibitor. Also retrieve the Sample Dissociation Tube from the
room temperature Consumables portion.
3. Place everything except for the Reducing
Agent B on ice and begin to prepare the buffers. After Reducing Agent B
thaws and everything is briefly vortexed, begin preparing the
buffers.
4. Make an appropriate amount of
buffer for the number of samples that you are going to be
isolating nuclei from
Lysis Buffer
Checkbox
Solution
PN
1X+10% (µl)
4X+10% (µl)
8X+10% (µl)
â–¡
Lysis Reagent
2000558
550.00
2200.0
4400.0
â–¡
Reducing Agent B
2000087
0.55
2.2
4.4
â–¡
Surfactant A
2000559
5.50
22.0
44.0
Total
556.05
2224.2
4448.4
Debris Removal
Buffer
Checkbox
Solution
PN
1X+10% (µl)
4X+10% (µl)
8X+10% (µl)
â–¡
Debris Removal Reagent
2000560
550.00
2200.0
4400.0
â–¡
Reducing Agent B
2000087
0.55
2.2
4.4
â–¡
Total
NA
550.55
2202.2
4404.4
Wash and Resuspension
Buffer
Checkbox
Solution
PN
1X+10% (µl)
4X+10% (µl)
8X+10% (µl)
â–¡
1X PBS (not provided)
NA
2887.5
11550
23100
â–¡
10% BSA (not provided)
NA
330.0
1320
2640
â–¡
RNase Inhibitor
2000565
82.5
330
660
Total
NA
3300.0
13200
26400
5. Keep all completed buffers labeled
and on ice. Pre-chill the centrifuge to
4°C.
Main Procedure
1. Retrieve Kidney Synthetic
Assembloids from the -80°C storage and place on ice and let it
thaw.
2. Add 100 µL of the Lysis
Buffer as soon as the tube is thawed.
3. Then using the green
pestle provided in the room-temperature pack, grind up the
tissue, pressing down and twisting 5 times (avoiding
the generation of bubbles) or until the solution is smooth with no
visible chunks.
4. Next, add 100 µL of the Lysis
buffer and pipette up and down, making sure that there are no
chunks.
5. Pre-coat round bottom FACS
tubes by pipetting 100 of 10% BSA into tube, swirl around and
then discard. Transfer the nuclei solution into the round bottom
FACS tube.
6. Spin the sample in a swinging
bucket centrifuge at 500 rcf for 3 min at 4°C.
7. Next, remove the
supernatant, being careful to avoid the pellet. OK to leave a
minimal volume of solution in the tube (~100 µL).
8. Re-suspend the pellet
with 500 µL of the Debris Removal Buffer. Spin at 700 rcf for 10 minutes
at 4°C.
9. Remove the supernatant
again and resuspend in 500 µL of the Wash and
Resuspension Buffer. Spin at 500 rcf for 5 minutes, 4 °C. Remove
supernatant.
10. Repeat step 9.
11. Finally, resuspend the. nuclei
pellet in 100 µL of the Final Wash and Resuspension Buffer.
Vortex the solution for 3 seconds and let it sit undisturbed for 15
minutes.
Now, depending on QC or following uses, use the concentrated
solution.
Expected Outcomes
In our hands, we started with ~400K nuclei from 5
Mature Kidney Synthetic Assembloids (quantified after initial
lysis)
After completing this protocol, we ended up with ~250K
nuclei in 100 µL at a concentration of 2,025
nuc/µL
This means that the each quadrant on the
hemocytometer had approcimately 20 healthy nuclei
Nuclei Count with Light Microscope:
With the nuclei solution, add 2 µL of nuclei to
18 µL of Trypan Blue, pipetting up and down into the TB
at least 10 times to mix thoroughly. Note that while Trypan Blue does
not accumulate in intact cells, the compound does concentrate within the
exposed nuclei, which is what we will be checking for.
Add 10 µL to one of the channels on a hemocytometer,
making sure the solution fills the space. Observe on a light microscope,
counting to get an approximation and assessment of nuclei quality.
From this procedure, the average expected nuclei concentration count
is: 500 nuclei per microliter This is approximately 20 to 40 nuclei per
quadrant in the hemocytometer.
Nuclei Quality Check:
Priority Check:
No clumps, with an even distribution
Quality:
A grade Nuclei: Good, circular shape with no
blebbing, halo of Trypan Blue around the edges that suggest an
undisturbed nuclear envelope. No aggregation.
B grade Nuclei: Slightly irregular shapes, still has
the halo but less bright or more uneven
C grade Nuclei: Losing more of normal shape, small
blebbing, paler color
D/F grade Nuclei: Complete loss of structure, no
blue halo, aggregates
Example of a graded sample:
Example of grading with reasoning. Note: this is an image of an
aggregate that was included because of a wide variety of quality, but as
a general rule aggregates indicate a low quality overall sample.
Example of a sample viewed at 10x and 40x, grading the individual
nuclei quality is easier at 40x but it is recommended to conduct overall
sample quality checks at 10x magnification.